1. Field of the Invention
This invention relates to aptamers and methods for the selection and generation of aptamers for use in such fields of application as proteomics, protein detection and purification, drug design and development, and protein purification and capture reagents.
2. Prior Relevant Art
The future success of proteomics depends on its ability to follow in the footsteps of genomics, where the development of new technologies generated an abundance of sequence data enabling researchers to probe problems that relate to the entire nucleic acid component of the cell. For the promise of proteomics to be realized, new tools are needed that will enable large-scale investigations of protein structure, function, and interactions. Significant progress has been made in proteomic technology development in many areas, including high-throughput gene cloning, protein production, mass spectrometry, 2-D PAGE, and microfluidics to allow large-scale proteomics to proceed.
One important set of tools that has been improved with moderate success are affinity reagents that function as antibodies to serve as protein probes. Affinity molecules that specifically bind proteins of interest can detect bound proteins in a protein microarray, or capture protein complexes for functional identification. Often these molecules can alter biological activity due to their binding and inhibit critical interactions by sterically blocking access to active sites and interaction surfaces, and thus present an opportunity to serve as functional probes as well as therapeutics. Traditionally, antibodies have satisfied the demand for such ligands, however as recombinant protein production gains throughput and pharmaceutical target repertoires expand, the ability to efficiently generate antibodies quickly falls short.
Several alternatives to antibodies have been investigated, such as single chain antibodies (scFv), peptides displayed on protein domain scaffolded surfaces, peptides, and peptoids (synthetic peptides). Each of these alternatives has drawbacks that limit their uses, such as problems of stability in varying conditions (ionic strength, temperature, and pH) and of low affinity, making some antibody alternatives ineffective for detecting proteins under many conditions.
The use of aptamers as protein affinity reagents offers advantages over the use of antibodies. Nucleic acids are easily synthesized or amplified by PCR; therefore a vast supply of consistent quality is available. Also, nucleic acids can easily be modified to incorporate tags, such as biotin or fluorescent molecules, for detection and/or immobilization. Additionally, aptamers are smaller (<25 kDa) and more stable than antibodies. Moreover, unlike the requirement of milligram quantities of protein or peptide for antibody production, only microgram quantities of protein or peptide are required for aptamer selection. These properties, coupled to the present technology available for DNA microarrays, make aptamers very suitable for use in protein microarrays as a ligand, or for detecting proteins bound to a chip surface (See Walter G, Bussow K, Lueking A, Glokler J. (2002) High-throughput protein arrays: prospects for molecular diagnostics. Trends Mol Med. June; 8(6):250-3.).
The idea of using single stranded nucleic acids (aptamers) as affinity molecules for proteins has shown modest progress. See Tuerk C, Gold L. (1990) Systematic evolution of ligands by exponential enrichment: RNA ligands to bacteriophage T4 DNA polymerase. Science. August 3;249(4968):505-10; Ellington A D, Szostak J W. (1990) In vitro selection of RNA molecules that bind specific ligands. Nature. August 30;346(6287):818-22; and Ellington A D, Szostak J W. (1992) Selection in vitro of single-stranded DNA molecules that fold into specific ligand-binding structures. Nature. February 27;355(6363):850-2. The concept is based on the ability of short oligomer (20-80 mer) sequences to fold, in the presence of a target, into unique 3-dimensional structures that bind the target with high affinity and specificity. Aptamers are generated by a process that combines combinatorial chemistry with in vitro evolution, commonly known as SELEX (Systematic Evolution of Ligands by Exponential Enrichment). Following the incubation of a protein with a library of DNA or RNA sequences (typically 1014 molecules in complexity) protein-DNA complexes are isolated, the DNA is amplified, and the process is repeated until the sample is enriched with sequences that display high affinity for the protein of interest. Since the selection pressure is high affinity for the target, aptamers with low nanomolar affinities may be obtained. Aptamers offer advantages over protein-based affinity reagents because nucleic acids possess increased stability, ease of regeneration (PCR or oligonucleotide synthesis), and simple modification for detection and immobilization.
Although SELEX appears to be technically very simple, small alterations to a protocol can have a large impact on the success of generating aptamers. Perhaps this explains why thirteen years since its first citation in the literature, only approximately forty protein-detecting aptamer sequences have been published, and very few have been well characterized. Although high-throughput methods for aptamer production have been published, most require expensive robotics and have not produced aptamers against large numbers of diverse targets (Cox J C, Rajendran M, Riedel T, Davidson E A, Sooter L J, Bayer T S, Schmitz-Brown M, Ellington A D. (2002) Automated acquisition of aptamer sequences. Comb Chem High Throughput Screen. June; 5(4):289-99).
Many variations in aptamer production protocols have been described in which the method of protein target partitioning seems to vary the most. Unbound DNA molecules have been removed from target proteins via: 1) filtration on a membrane (Ellington A D, Szostak J W. (1992) Selection in vitro of single-stranded DNA molecules that fold into specific ligand-binding structures. Nature. February 27;355(6363):850-2); 2) column chromatography, in which the targets are bound to a matrix, such as sepharose, using a covalent linkage or an affinity tag (Ylera F, Lurz R, Erdmann V A, Furste J P. (2002) Selection of RNA aptamers to the Alzheimer's disease amyloid peptide. Biochem Biophys Res Commun. February 8;290(5):1583-); and 3) binding of the protein to the wells of a microtiter plate (Drolet D W, Jenison R D, Smith D E, Pratt D, Hicke B J. (1999) A high throughput platform for systematic evolution of ligands by exponential enrichment (SELEX). Comb Chem High Throughput Screen. October; 2(5):271-8).
Gorenstein, et al, in U.S. Pat. No. 6,423,493, describe a random combinatorial selection method for the construction of oligonucleotide aptamers in which nuclease resistance is conferred by the inclusion of modified nucleotides. The modified nucleotides are incorporated during PCR amplification to form achiral modified oligonucleotides. Thio-substituted aptamers are provided that bind tightly to the nuclear factor for human IL6 (NF-IL6).
Kwagh, et al., in U.S. Pat. No. 6,515,120, describe a method for sequencing and structurally characterizing a polymeric biomolecule using aptamers and also describes aptamers that recognize and bind to AMP, dAMP, GMP, dGMP, CMP and dCMP.
In U.S. Pat. No. 6,180,348, Li describes a method that makes use of magnetic separation to identify an aptamer which specifically binds to a target molecule of interest. A method for identifying oligomer sequences, optionally comprising modified bases, which specifically bind target molecules such as serum proteins, kinins, eicosanoids and extracellular proteins is described by Griffin, et al in U.S. Pat. No. 5,756,291. The method is used to generate aptamers that bind to serum Factor X, PDGF, FGF, ICAM, VCAM, E-selectin, thrombin, bradykinin, PGF2 and cell surface molecules.